Biofilms are medically and industrially important because they can accumulate on a wide variety of substrates and are resistant to antimicrobial agents and detergents. Microbial biofilms develop when microorganisms adhere to a surface and produce extracellular polymers that facilitate adhesion and provide a structural matrix. Therefore inhibiting adhesion to surfaces is important. This surface may be inert, non-living material or living tissue.
Biofilm-associated microorganisms behave differently from planktonic (freely suspended) organisms with respect to growth rates and ability to resist antimicrobial treatments and therefore pose a public health problem. Many chronic infections that are difficult or impossible to eliminate with conventional antibiotic therapies are known to involve biofilms. A partial list of the infections that involve biofilms includes: otitis media, prostatitis, vascular endocarditis, cystic fibrosis pneumonia, meliodosis, necrotising faciitis, osteomyelitis, peridontitis, biliary tract infection, struvite kidney stone and host of nosocomial infections (Costerton, J. W., et al., Science, 284:1318–1322, 1999).
Biofilms on indwelling medical devices may be composed of gram-positive or gram-negative bacteria or yeasts. Bacteria commonly isolated from these devices include the gram-positive Enterococcus faecalis (E. faecalis), Staphylococcus epidermidis (S. epidermidis), Staphylococcus aureus (S. aureus), Streptococcus viridans (St. viridans); and the gram-negative Escherichia coli (E. coli, Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabilis) and Pseudomonas aeruginosa (P. aeruginosa) (Donlan, R. M., Emerging Infectious Diseases, 7:277–281, 2001). The organisms most commonly isolated from urinary catheter biofilms are Staphylococcus epidermidis, Enterococcus faecalis, E. coli, Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. In the case of vascular catheters, Staphylococcus aureus and Staphylococcus epidermidis account for almost 70–80% of all infectious organisms, with Staphylococcus epidermidis being the most common organism. Candida albicans accounts for about 10–15% of catheter infections. Gram-negative bacilli account for almost 60–70%, enterococci for about 25% and Candida albicans for about 10% of cases of urinary tract infections. Catheter-associated urinary tract infection is the most common nosocomial infection. Each year, about 1 million patients in US hospitals acquire such an infection. It is the second most common cause of nosocomial infections (Maki, D. G. and P. A. Tambyah, Emerging Infectious Diseases, 7:1–6, 2001).
In recent years, there have been numerous efforts to sequester antimicrobials and antibiotics on the surface of or within devices that are then placed in the vasculature or urinary tract as a means of reducing the incidence of device-related infections. These antimicrobial agents are of varying chemical composition and cationic polypeptides (protamine, polylysine, lysozyme, etc.), surfactants (SDS, Tween-80, surfactin, etc.), quaternary ammonium compounds (benzalkonium chloride, tridodecyl methyl ammonium chloride, didecyl dimethyl ammonium chloride, etc.). The iron-sequestering glycoproteins such as lactoferrin from milk and ovotransferrin (conalbumin) from egg white are iron-binding glycoproteins, which inhibit the growth of certain bacteria by making iron unavailable for bacterial metabolism (Bezkorovainy, A., Adv. Exp. Med. Biol. 135:139–154, 1981).
The main methods of antimicrobial catheter preparation include immersion or flushing, coating, drug-polymer conjugate and impregnating (Tunny, M. M., et al., Rev. Med. Microbiol., 74: 195–205, 1996). In a clinical setting, suitable catheters can be treated by immersion immediately prior to placement, which offers flexibility and control to clinicians in certain situations. Several studies have examined the clinical efficacy of catheters coated with antimicrobial agents. Minocycline and rifampin coatings have been shown to significantly reduce the risk of catheter-associated infections (Raad, I. I. et al., Crit. Care Med., 26: 219–224, 1998). Minocycline coated onto urethral catheters has been shown to provide some protection against colonization (Darouiche, R. O., et al., Int. J. Antimicrob. Ag. 8: 243–247, 1997). Johnson, et al., described substantial in vitro antimicrobial activity of a commercially available nitrofurazone coated silicone catheter in comparison with commercial silver-hydrogel coated catheter (Antimicrob. Agents. Chemother. 43: 2,990–2,995, 1999). The antibacterial activity of silver-containing compounds as antimicrobial coatings for medical devices has been widely investigated. Silver-sulfadiazine used in combination with chlorhexidine has received particular interest as a central venous catheter coating (Stickler, D. J., Curr. Opin. Infect. Dis., 13:389–393, 2000; Darouiche, R. O., et al., New Eng. J. Med., 340:1–8, 1999). Pugach, et al., explored in vivo efficacy of liposomal hydrogel coated urinary catheters for the prevention of bacterial biofilms on the external catheter surface (J. Urol. 162: 883–887, 1999).
The loading of antimicrobial agents into medical devices by immersion or coating technologies has the advantage of being relatively simple. However, the limited mass of drug that can be incorporated may be insufficient for a prolonged antimicrobial effect, and the release of the drug following clinical insertion of the device is rapid and relatively uncontrolled. A means of reducing these problems is by direct incorporation of the antimicrobial agent into the polymeric matrix of the medical device at the polymer synthesis stage or at the device manufacture stage. Rifampicin has been incorporated into silicone in an attempt to prevent infection of cerebrospinal fluid shunts with some success (Schierholz, J. M., et al., Biomaterials, 18: 839–844, 1997). Iodine has also been incorporated into medical device biomaterials. Coronary stents have been modified to have antithrombogenic and antibacterial activity by covalent attachment of heparin to silicone with subsequent entrapment of antibiotics in cross-linked collagen bound to the heparinised surface (Faligren, C., et al., Zentralbl. Bakteriol., 287:19–31, 1998).
Welle, C. J., et al., in U.S. Pat. No. 6,187,768 disclosed the method of preparing a kit for flushing a medical device. The kit includes a solution containing an antibiotic, an anticoagulant (protamine sulfate) and an antithrombotic agent or chelating agent useful for preventing infections caused by bacterial growth in catheters. Budny, J. A. et al., discloses various antimicrobial agents for anchoring to biofilms (US Patent Application No. 20020037260). Raad, et al., in U.S. Pat. No. 5,362,754 disclosed that pharmaceutical compositions of a mixture of minocycline and EDTA were useful in maintaining the patency of a catheter port. U.S. Pat. No. 6,187,768 to Welle et al. teaches the use of several anticoagulants for use in medical devices, including protamine sulfate.
In medical devices, various techniques have been described that incorporate potentially toxic metal ions in the form of metal salts into materials that make up the medical devices. The protection against biofilm formation lasts only as long as the coating remains on the device. A method of long-term prevention from biofilm formation that acts at the level of prevention of biofilm formation is needed. Also needed is a composition that allows for low quantities of the composition to be used effectively, thus reducing toxicity or other side effects to the user or patient without sacrificing effectiveness against biofilm formation. There is also a need for compositions that are environmentally friendly, medically acceptable, effective at lower concentrations and relatively economical to manufacture on a commercial scale for reducing biofilm formation in biomedical devices.
A few recent studies have demonstrated the antimicrobial activity of thiol-specific reagents, such as, N-substituted maleimides and thiosulfinates (Cechinel Filho, V., et al., Farmaco. 49: 675–677, 1994; Yoshida, H., et al., Biosci. Biotechnol. Biochem. 63: 591–594, 1999 and Zentz, F., et al., Farmaco. 57: 21–426, 2002). Wu, et al., in U.S. Pat. No. 5,466,707, disclosed the use of thione maleimides and compositions containing them as antimicrobial and marine antifouling agents. Thiol-specific reagents react with thiol groups of various enzymes, such as, thioredoxin reductase, coenzyme A disulfide reductase and glucosamine-1-phosphate acetyltransferase in bacteria (Ankri, S. and D. Mirelman, Microbes Infect. 1: 125–129, 1999; Delcardayre and Davies, International Publication No. WO 97/23628 and Pompeo, F. et al., J. Bacteriol. 180: 4799–4803, 1998). US Patent Application No. 20030166843 from Benson, T. E. describes the use of x-ray crystal structure for solving the structure of S. aureus thioredoxin reductase and other molecular complexes, and designing inhibitors of Staph. aureus thioredoxin reductase. DeBouck, et al., in U.S. Pat. No. 6,043,071, described the methods for utilizing glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase (GImU) polypeptides to screen for antibacterial compounds. Donna et al. observed 95% decrease in thioredoxin reductase activity when it was crosslinked with thiol-specific reagent N,N′-(1,2-phenylene) dimaleimide (Prot. Sci. 7:369–375, 1998). The structural differences between the bacterial and mammalian thioredoxin reductases and a surprising diversity in their chemical mechanism of thioredoxin reduction suggest that it could be used as a target for the development of new antimicrobials (Becker, K. et al., Eur. J. Biochem. 267: 6118–6125, 2000 and Uziel, O. et al., J. Bacteriol. 186: 326–334, 2004).